Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 100 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis. However, the Center for Disease Control does not presently recommend immunoglobulins; for this purpose.
The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions.
Various clinical studies have been conducted with the goal of identifying pharmaceutical agents capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment.
Until recently, interferon (IFN) was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C. However the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients. Recently, interferon in combination with ribavirin has been approved for patients non-responsive to IFN alone. However, the side effects caused by IFN are not alleviated with this combination therapy.
Therefore, a need exists for the development of effective antiviral agents for treatment of HCV infection that overcomes the limitations of existing pharmaceutical therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV.
A general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus. In this vein, patent application WO 97/06804 describes the (-) enantiomer of the nucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) as active against HCV. This compound, although reported as safe in previous clinical trials against HIV and HBV, has yet to be clinically proven active against HCV and its mechanism of action against the virus has yet to be reported.
Intense efforts to discover compounds which inhibit the NS3 protease or RNA helicase of HCV have led to the following disclosures:
U.S. Pat. No. 5,633,388 describes heterocyclic-substituted carboxamides and analogues as being active against HCV. These compounds are directed against the helicase activity of the NS3 protein of the virus but clinical tests have not yet been reported. PA0 A phenanthrenequinone has been reported by Chu et al (Tet. Lett., (1996), 7229-7232) to have activity against the HCV NS3 protease in vitro. No further development on this compound has been reported. PA0 A paper presented at the Ninth International Conference on Antiviral Research, Urabandai, Fukyshima, Japan (1996) (Antiviral Research, 30, 1, 1996; A23 (abstract 19)) reports thiazolidine derivatives to be inhibitory to the HCV protease. PA0 WO 98/17679 from Vertex Pharmaceuticals Inc. discloses inhibitors of serine protease, particularly, Hepatitis C virus NS3 protease. These inhibitors are peptide analogues based on the NS5A/5B natural substrate that contain C-terminal aldehydes, .alpha.-ketoamides and fluorinated ketones. PA0 Hoffman LaRoche has also reported hexapeptides that are proteinase inhibitors useful as antiviral agents for the treatment of HCV infection. These peptides contain an aldehyde or a boronic acid at the C-terminus. PA0 Steinkuhler et al. and Ingallinella et al. have published on NS4A-4B product inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914). However, these peptides and analogues were published after the priority date of the present application. PA0 a is 0 or 1; PA0 R.sub.6, when present, is carboxy(lower)alkyl; PA0 b is 0 or 1; PA0 R.sub.5, when present, is C.sub.1-6 alkyl, or carboxy (lower) alkyl; PA0 Y is H or C.sub.1-6 alkyl; PA0 R.sub.4 is C.sub.1-10 alkyl; cycloalkyl C.sub.3-10 ; PA0 R.sub.3 is C.sub.1-6 alkyl; cycloalkyl C.sub.3-10 ; PA0 W is a group of formula II: ##STR3## wherein R.sub.2 is C.sub.1-10 alkyl or C.sub.3-7 cycloalkyl optionally substituted with carboxyl; C.sub.6 or C.sub.10 aryl; or C.sub.7-16 aralkyl; or PA0 W is a group of formula II': ##STR4## wherein X is CH or N; and R.sub.2 ' is C.sub.3-4 alkylene that joins X to form a 5- or 6-membered ring, said ring optionally substituted with OH; SH; NH.sub.2 ; carboxyl; R.sub.12, OR.sub.12, SR.sub.12, NHR.sub.12 or NR.sub.12 R.sub.12 ' PA0 Q is a group of the formula: ##STR5## wherein Z is CH or N; X is O or S; PA0 R.sub.1 is H, C.sub.1-6 alkyl or C.sub.1-6 alkenyl both optionally substituted with thio or halo; and PA0 when Z is CH, then R.sub.13 is H; CF.sub.3 ; CF.sub.2 CF.sub.3 ; CH.sub.2 --R.sub.14 ; PA0 CH (F)--R.sub.14 ; CF.sub.2 --R.sub.14 ; NR.sub.14 R.sub.14 ' S--R.sub.14 ; or CO--NH--R.sub.14 wherein PA0 Q is a phosphonate group of the formula: ##STR6## wherein R.sub.15 and R.sub.16 are independently C.sub.6-20 aryloxy; and R.sub.1 is as defined above. PA0 C.sub.1-6 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyloxy or C.sub.1-6 alkoxy; PA0 C.sub.3-7 cycloalkyl optionally substituted with carboxyl, MeOC(O), EtOC(O) or BnOC(O); PA0 3-carboxypropionyl (DAD) or 4-carboxybutyryl (DAE); or ##STR11## PA0 i) For the synthesis of trifluoromethyl alcohols of formula Vd the procedure described by J. W. Skiles et al. (J. Med. Chem. (1992), 35, 641-662) was used as illustrated: ##STR32## wherein R.sub.1 is as defined above. PA0 ii) For the synthesis of pentafluoroethyl alcohols of formula VId, the procedure described by M. R. Angelastro et al. (J. Med. Chem., (1994), 37, 4538-4554) was used as illustrated in scheme VI: ##STR33## wherein R.sub.1 is as defined above. PA0 iii) For the synthesis of hydroxy amides of formula VIIf the procedure described by Peet et al. (Tet. Lett. (1988), 3433) was used as illustrated in scheme VII: ##STR34## wherein R.sub.1 is as defined above. PA0 iv) The coupling to P2-P6 was carried out as illustrated in scheme VIII: ##STR35## a) The P6 to P2 fragment can be linked to the free amino group of the amino alcohol derivative VIIIa as described previously in Scheme I to give the peptido alcohol VIIIb. PA0 b) The alcohol functionality of the peptido alcohol VIIb is then oxidized by techniques and procedures well known and appreciated by one of ordinary skill in the art, such as the Swern Oxidation (Tidwell, T. T., Synthesis, (1990), 857-870), or more specifically the Pfitzner-Moffatt oxidation (K. E. Pfitzner, and J. G. Moffatt, J. Am. Chem. Soc., (1965), 5670-5678) and the Dess-Martin periodinane method (D. B. Dess or J. C. Martin, (J. Org. Chem., (1983), 48, 4155-4156) to give the compounds of formula I wherein Q contains an activated carbonyl. PA0 D) Synthesis of compounds of formula I wherein Q is: ##STR36## wherein Z is N; and R.sub.13 is NHR.sub.14, NR.sub.14 R.sub.14 ', CH.sub.2 --R.sub.14, CHR.sub.14 R.sub.14 ' or O--R.sub.14 ; wherein R.sub.14 and R.sub.1 are as defined above, was done as described in scheme X. PA0 i) For the synthesis of the aza-containing P1 fragments, the procedure described by A. S. Dutta et al. (J. Chem. Soc. Perkin I, (1975), 1712) was followed as illustrated: ##STR37## wherein R.sub.1 and R.sub.14 are as defined hereinabove. PA0 ii) Coupling of P2-P6 was carried out according to scheme X: ##STR38## wherein z is O, NH, CH.sub.2, CHR.sub.14', or NR.sub.14'. PA0 E) Synthesis of compounds of formula I wherein Q is: ##STR39## wherein R.sub.15 and R.sub.16 are as defined above, the procedure described by J. Oleksyszyn et al. (Synthesis, (1979), 985-1386) was used as illustrated in scheme XI: ##STR40## wherein R.sub.1 is as defined hereinabove.
Several studies have reported compounds inhibitory to other serine proteases, such as human leukocyte elastase. One family of these compounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). The peptides disclosed in that application are morpholinylcarbonyl-benzoyl-peptide analogues that are structurally different from the peptides of the present invention.
One advantage of the present invention is that it provides peptides that are inhibitory to the NS3 protease of the hepatitis C virus.